ZIKV NS2B/NS3 protease expression, purification and enzyme inhibition assay

To express ZIKV NS2B/NS3 protease, the gene of ZIKV NS3 protease (residues 1–170) connected with the NS2B core region (residues 49–95) by a GGGGSGGGG linker was codon-optimized, synthesized (GenScript) and inserted into an expression plasmid vector pET28a pET28a using NcoI and XhoI restriction enzyme cutting sites. The recombinant plasmid was transfected into Rosetta2(DE3)pLysS (Novagen) cells and the clones positive for the plasmid were cultured overnight to an OD600 of 0.8 at 37 °C in LB medium and were then induced with 1 mM IPTG for 5 hours at 25 °C. The cells were harvested by centrifugation at 4 °C (6000 rpm, 10 min) followed by lysis by sonication in lysis buffer containing 1X PBS (137 mM NaCl, 10 mM Phosphate, 2.7 mM KCl, pH 7.4), 0.1% Triton X100 and 20 mM imidazole. The His-tagged ZIKV protease was purified by Ni Sepharose Fast Flow column (GE Healthcare) with a stepwise gradient of purification buffer (20 mM, 40 mM, 100 mM, 500 mM imidazole in 1X PBS). The purified ZIKV NS2B/NS3 protease was analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (10%), was aliquoted with approximately 95% purity and stored at −20 °C.

The protease activity tests were performed in a 96-well black flat-bottomed microtiter plates (Greiner Bio one, Germany) with a final volume of 100 µl. ZIKV NS2B/NS3 recombinant protease, at a final concentration of 5 nM, was pre-incubated for 15 min at 37 °C with either Asunaprevir and Simeprevir at different concentrations in the assay buffer (20 mM Tris pH7.5, 0.05% CHAPS, 1% Glycerol). The FRET substrate Benzoyl–Nle–KKR–AMC substrate was then added at a final concentration of 20 µM to the enzymatic reaction incubated for 30 min at 37 °C. The data for the same compound concentrations with the substrate and without enzyme were also measured as a control. The fluorescence signals (excitation/emission: 355 nm/460 nm) of the released AMC was measured using a fluorometer (VICTOR2, PerkinElmer). The results were plotted as dose-inhibition curves using non-linear regression with a variable slope to determine the IC50 values of inhibitor compounds (with GraphPad Prism V6.0). All assays were performed in duplicates in 96-well plates.